Peptides for use in immunotherapy of autoimmune diseases

ABSTRACT

The invention relates to the use of novel peptides in a peptide induced tolerance therapy to prevent autoimmune disorders and in particular their use in treatment of chronic destruction of articular cartilage. The invention furtermore embraces pharmaceutical compositions comprising said peptides and a diagnostic method for the detection of autoreactive T cells in a test sample.

This application is a 35 U.S.C. 371 National Phase application ofPCT/EP99/05050 filed Jul. 16, 1999, which is based for priority onEuropean Application EP 98202470.5 filed Jul. 23, 1998.

The invention relates to novel peptides, their use in treatment ofchronic destruction of articular cartilage in autoimmune diseases,pharmaceutical compositions comprising said peptide, and a diagnosticmethod for the detection of autoreactive T cells in a test sample.

The immune system is established on a principle of discriminationbetween foreign antigens (non-self antigens) and autoantigens (selfantigens, derived from the individuals own body) achieved by a build-intolerance against the autoantigens.

The immune system protects individuals against foreign antigens andresponds to exposure to a foreign antigen by activating specific cellssuch as T- and B lymphocytes and producing soluble factors likeinterleukins, antibodies and complement factors. The antigen to whichthe immune system responds is degraded by the antigen presenting cells(APCs) and a fragment of the antigen is expressed on the cell surfaceassociated with a major histocompatibility complex (MHC) class IIglycoprotein. The MHC-glycoprotein-antigen-fragment complex is presentedto a T cell which by virtue of its T cell receptor recognizes theantigen fragment conjointly with the MHC class II protein to which it isbound. The T cell becomes activated, i.e. proliferates and/or producesinterleukins, resulting in the expansion of the activated lymphocytesdirected to the antigen under attack (Grey et al., Sci. Am., 261:38-46,1989).

Self antigens are also continuously processed and presented as antigenfragments by the MHC glycoproteins to T cells (Jardetsky et al., Nature353:326 -329, 1991). Self recognition thus is intrinsic to the immunesystem. Under normal circumstances the immune system is tolerant to selfantigens and activation of the immune response by these self antigens isavoided.

When tolerance to self antigens is lost, the immune system may becomeactivated against one or more self antigens, resulting in the activationof autoreactive T cells and the production of autoantibodies. Thisphenomenon is referred to as autoimmunity. As the immune response ingeneral is destructive, i.e. meant to destroy the invasive foreignantigen, autoimmune responses can cause destruction of the body's owntissue.

The contribution of T cells to autoimmune diseases has been establishedin several studies. In mice, experimental autoimmune encephalomyelitis(EAE) is mediated by a highly restricted group of T cells, linked bytheir specificity for a single epitope of myelin basic protein (MBP)complexed to an MHC class II molecule. In the Lewis rat, a species withhigh susceptibility to various autoimmune diseases, disease has beenshown to be mediated by T cells. In humans autoimmune diseases are alsothought to be associated with the development of auto-aggressive Tcells.

A destructive autoimmune response has been implicated in variousdiseases such as rheumatoid arthritis (RA), in which the integrity ofarticular cartilage is destroyed by a chronic inflammatory processresulting from the presence of large numbers of activated lymphocytesand MHC class II expressing cells. The mere presence of cartilageappears necessary for sustaining the local inflammatory response: it hasbeen suggested that cartilage degradation is associated with theactivity of cartilage-responsive autoreactive T cells in RA (Sigall etal., Clin. Exp. Rheumat. 6:59, 1988; Glant et al., Biochem. Soc. Trans.18:796, 1990; Burmester et al., Rheumatoid artritis Smolen, Kalden,Maini (Eds) Springer-Verlag Berlin Heidelberg, 1992). Furthermore,removal of cartilage from RA patients by surgery was shown to reduce theinflammatory process (R. S. Laskin, J. Bone Joint Surgery (Am) 72:529,1990). The cartilage proteins are therefore considered to be targetautoantigens which are competent of stimulating T cells. Activation ofthese autoreactive T cells leads to development of autoimmune disease.However, the identification of the autoantigenic components that play arole in the onset of rheumatoid arthritis has so far remained elusive.

The inflammatory response resulting in the destruction of the cartilagecan be treated by several drugs, such as for example steroid drugs.However, these drugs are often immunosuppressive drugs that arenonspecific and have toxic side effects. The disadvantages ofnonspecific immunosuppression makes this a highly unfavourable therapy.

The antigen-specific, nontoxic immunosuppression therapy provides a veryattractive alternative for the nonspecific immunosuppression. Thisantigen-specific therapy involves the treatment of patients with thetarget autoantigen or with synthetic T cell-reactive peptides derivedfrom the autoantigen. These synthetic peptides correspond to T cellepitopes of the autoantigen and can be used to induce specific T celltolerance both to themselves and to the autoantigen. Although it seemsparadoxical to desensitize the immune system with the very same antigenresponsible for activating the immune system, the controlledadminstration of the target (auto)antigen can be very effective indesensitization of the immune system. Desensitization or immunologicaltolerance of the immune system is based on the long-observed phenomenonthat animals which have been fed or have inhaled an antigen or epitopeare less capable of developing a systemic immune response towards saidantigen or epitope when said antigen or epitope is introduced via asystemic route.

The human cartilage glycoprotein-39 (HC gp-39) was previously identifiedas a target autoantigen in rheumatoid arthritis (RA) (Verheijden et al.,Arthitis Rheum. 40:1115-1125, 1997). The strategy followed foridentification of relevant auto-epitopes within HC gp-39 was based onthe assumption that the DR4 or DR1 molecules predispose to RA (Gao etal., Arthitis Rheum. 33:939-946, 1990; Nelson et al., RheumatoidArthritis, In Proceedings of the Eleventh InternationalHistocompatibility Workshop and Conference. Vol 1, Tsuji et al Ed,Oxford University Press, 1991) at two levels, firstly, by shaping the Tcell repertoire and secondly, by determinant selection. The sharedepitope found among the RA-associated DR molecules might be involved inselection of similar sets of peptides for presentation to T cells(Gregerson et al., Arthitis Rheum. 30:1205-1213, 1987). Putative bindingsequences within the primary structure of HC gp-39 were identified byuse of a DR4 (B1*0401) peptide binding motif (Verheijden et al.,Arthitis Rheum. 40:1115-1125, 1997). HC gp-39, a protein of 362 aminoacids, excluding the signal sequence (Hakala et al., J. Biol. Chem.268:25803-25810, 1993), contains six regions accommodating this motif.Four peptides thus selected were synthesized and tested for binding theRA-associated DR1 and DR4 (B1*0401 and 0404) variants. All motif-basedpeptides, spanning residues 103-116, 259-275, 263-275 and 326-338 of HCgp-39, were found to bind with high relative affinity to DRB1*0401molecules. The recognition of these peptides by peripheral blood T cellsfrom RA patients and healthy donors was subsequently examined. Allmotif-based peptides were readily recognized in RA patients, therebysuggesting a high frequency of HC gp-39-specific T cells in RA. Theresponse to 263-275 was most prominent; 8 out of 18 RA patientsresponded to this peptide (Verheijden et al., Arthitis Rheum.40:1115-1125, 1997). Thus, HC gp-39 is a target for immune recognitionin the joint.

The significance of this protein for arthritic disease was furtherdemonstrated by its arthritogenicity in Balb/c mice. A single injectionin the chest region with μg amounts of protein mixed in IFA, induced achronic joint inflammation reminiscent of RA (Verheijden et al.,Arthitis Rheum 40:1115-1125, 1997).

Recently, a novel human chondrocyte protein, YKL-39, was isolated anddescribed (Hu et al., J.Biol.Chem. 271: 19415-19420, 1996). The proteinshares significant sequence identity with HC gp-39 (YKL-40). Anotherhomologue of HC gp-39 is secreted by human macrophages and is termedchitotriosidase (Boot et al., J.Biol.Chem. 270: 26252-26256, 1995). Thesequences corresponding to the HC gp-39 (263-275) peptide RSFTLASSETGVG(SEQ ID NO:3) are identified as HSFTLASAETTVG (SEQ ID NO:2) within theYKL-39 protein (266-278) and as RSFTLASSSDTRVG (SEQ ID NO:4) withinmacrophage chitotriosidase (269-282) respectively (Table 1).

The chitotriosidase peptide Chi (269-282) contains the DRB1*0401,peptide binding motif which was previously used for selection of T-cellepitopes within proteins. In contrast, the YKL-39 (266-278) peptide doesnot contain this 0401 motif.

It will be clear that tolerization of HC gp-39 (263-275)-reactiveT-cells may be of benefit to RA patients. Likewise, mimicry epitopes ofHC gp39 (263-275) may have a similar function and may be used to inducetolerance. Preferably such mimicry epitopes will have at least the sametolerizing capacity.

To effectively use tolerance induction therapy to treat T cell mediatedcartilage destruction, there is a great need to identify T cell-reactivepeptides which can desensitize patients against the autoantigen that isactivating the T cells responsible for the inflammatory process.

Although the YKL-39 peptide does not contain the 0401 motif, it wassurprisingly found that the YKL-39 (266-278) epitope is a mimicryepitope of HC gp-39 (263-275).

This epitope therefore is useful for tolerization of autoreactiveT-cells with reactivity to HC gp-39 (263-275), YKL-39 (266-278) or theirmimicry epitopes in rheumatoid arthritis patients.

It is an object of the invention to provide peptides which are able toinduce systemic immunological tolerance, more in particular specific Tcell tolerance, preferably to the responsible cartilage antigen inpatients suffering from T cell-mediated cartilage destruction. Thepeptides of the present invention are characterized in that theycomprise one or more of the amino acid sequences FTLASAETT (SEQ ID NO:1). More specifically, a peptide according to the invention comprisesHSFTLASAETTVG (SEQ ID NO: 2).

Also within the scope of the invention are multimers of the peptidesaccording to the invention such as for example a dimer or trimer of thepeptides according to the invention. A multimer according to theinvention can either be a homomer, consisting of a multitude of the samepeptide, or a heteromer consisting of different peptides.

The characteristic amino acid sequences of the peptides according to theinvention can be flanked by random amino acid sequences. Preferred areflanking sequences, that have a stabilizing effect on the peptides, thusincreasing their biological availability.

Human Cartilage glycoprotein 39 is a target autoantigen in RA patientswhich activates specific T cells, thus causing or mediating theinflammatory process. HC gp-39 derived peptides were predominantlyrecognized by autoreactive T cells from RA patients but rarely by Tcells from healthy donors, thus indicating that HC gp-39 is anautoantigen in RA. The arthritogenic nature of HC gp-39 was furthersubstantiated in the Balb/c mouse. A single, subcutaneous injection ofsaid protein in Balb/c mice was able to initiate arthritic signs in theanimals. The course of the HC gp-39- induced disease was characterizedby relapses occuring periodically in fore paws and/or hind paws andgradually developed from a mild arthritis into a more severe form. Also,a symmetrical distribution of afflicted joints was observed which is,together with the observation of recurrent relapses, reminiscent ofdisease progression in arthritis, especially RA.

It was surprisingly found that the YKL-39 266-278 peptide was effectiveas a tolerogen. It will be clear to those skilled in the art that thepeptides may be extended at either side of the peptide or at both sidesand still exert the same immunological function. The extended part maybe an amino acid sequence similar to the natural sequence of the proteinYKL-39.

The peptides according to the invention can be prepared by well knownorganic chemical methods for peptide synthesis such as, for example,solid-phase peptide synthesis described for instance in J. Amer. Chem.Soc. 85:2149 (1963) and Int J. Peptide Protein Res. 35:161-214 (1990).The peptides according to the invention can also be prepared byrecombinant DNA techniques. A nucleic acid sequence coding for a peptideaccording to the invention or a multimer of said peptides is insertedinto an expression vector. Suitable expression vectors comprise thenecessary control regions for replication and expression. The expressionvector can be brought to expression in a host cell. Suitable host cellsare, for instance, bacteria, yeast cells and mammalian cells. Suchtechniques are well known in the art, see for instance Sambrooke et al,Molecular Cloning:a Laboratory Manual, Cold Spring Harbor laboratoryPress, Cold Spring Harbor, 1989.

The peptides may be stabilised by C- and/or N- terminal modifications,which will decrease exopeptidase catalysed hydrolysis. The modificationsmay include: C-terminal acylation, (e.g acetylation =Ac-peptide),N-terminal amide introduction, (e.g. peptide-NH₂) combinations ofacylation and amide introduction (e.g. Ac-peptide-NH₂) and introductionof D-amino acids instead of L-amino acids (Powell et al., J. Pharm Sci.,8:731-735, 1992).

Other modifications are focussed on the prevention of hydrolysis byendopeptidases. Examples of these modifications are: introduction ofD-amino acids instead of L-amino acids, modified amino acids,cyclisation within the peptide, introduction of modified peptide bonds,e.g. reduced peptide bonds ψ[CH₂NH] and e.g. peptoids (N-alkylatedglycine derivatives) (Adang et al, Recl. Trav. Chim. Pays-Bas,113:63-78, 1994 and Simon et al, Proc. Natl. Acad. Sci. USA,89:9367-9371, 1992).

The peptides according to the invention are T-cell epitopes, which arerecognized by and are able to stimulate autoreactive T-cells. Theseautoreactive T cells may be found e.g. in the blood of patientssuffering from autoimmune diseases.

Thus, according to the invention the peptides, said peptides resemblingthe MHC Class II restricted T-cell epitopes present on the targetautoantigen comprising the peptide of SEQ ID NO:1 or SEQ ID NO:2, arevery suitable for use in a therapy to induce specific T-cell toleranceto said autoantigen in mammals, more specifically humans, suffering fromT-cell mediated cartilage destruction, such as for example arthritis,more specifically rheumatoid arhritis. Optionally such a treatment canbe combined with the the administration of other medicaments such asDMARDs (Disease Modifying Anti-Rheumatic Drugs e.g. sulfasalazine,anti-malarials (chloroquine, hydroxychloroquine) injectable or oralgold, methotrexate, D-penicillamine, azathioprine, cyclosporine,mycophenolate), NSAIDs (non steroidal anti inflammatory drugs),corticosteroids or other drugs knowns to influence the course of thedisease in autoimmune patients.

The peptides according to the invention can also be used to modulatelymphocytes that are reactive to antigens other than said autoantigenbut are present in the same tissue as the autoantigen i.e. proteins orparts thereof comprising the peptide according to SEQ ID NO:1 or SEQ IDNO:2. By the induction of antigen-specific T-cell tolerance, autoimmnunedisorders can be treated by bystander suppression. More in general, thecells to be modulated are hematopoietic cells. In general, in order tofuction as a tolerogen the peptide must fulfill at least two conditionsi.e. it must possess an immune modulating capacity and it must beexpressed locally usually as part of a larger protein.

Thus, the present invention provides a method to treat patientssuffering from inflammatory autoimmune diseases, by administration of apharmaceutical preparation comprising the peptide according to theinvention. Such patients may suffer from diseases like Graves' diseases,juvenile arthritis, primary glomerulonephritis, osteoarthritis,Sjögren's syndrome, myasthenia gravis, rheumatoid arthritis, Addison'sdisease, primary biliary sclerosis, uveitis, systemic lupuserythematosis, inflammatory bowel disease, multiple sclerosis ordiabetes. The peptides according to the present invention therefore canbe used in the preparation of a pharmaceutical to induce tolerance inpatients suffering from these diseases.

Treatment of autoimmune disorders with the peptides according to theinvention makes use of the fact that bystander suppression is induced tounrelated but co-localized antigens. The regulatory cells secrete in anantigen specific fashion pleiotropic proteins such as cytokines whichmay downmodulate the immune response.

According to the invention, patients suffering from T-cell mediateddestruction of the articular cartilage can be treated with atherapeutical composition comprising one or more peptides according tothe invention and a pharmaceutical acceptable carrier. Administration ofthe pharmaceutical composition according to the invention will inducesystemic immunological tolerance, in particular tolerance of thespecific autoreactive T cells of these patients, to the autoantigenicproteins in the articular cartilage under attack and other self antigenswhich display the identified MHC Class II binding T cell epitopescharacterized or mimicked by the amino acid sequences of one or more ofthe peptides according to the invention. The induced tolerance thus willlead to a reduction of the local inflammatory response in the articularcartilage under attack.

Very suitable peptides to be used in a pharmaceutical compositionaccording to the invention are the peptides comprising the YKL-39(268-276) or the YKL-39 (266-278) peptide flanked by sequences up to atotal length of 55 amino acids. More preferably the peptides have alength of 25 amino acids. Even more preferably the amino acid sequenceof the peptides is FTLASAETT (SEQ ID NO: 1) or HSFTLASAETTVG (SEQ ID NO:2).

The peptides according to the invention have the advantage that theyhave a specific effect on the autoreactive T cells thus leaving theother components of the immune system intact as compared to thenonspecific suppressive effect of immunosuppressive drugs. Treatmentwith the peptides according to the invention will be safe and no toxicside effects will occur.

Systemic immunological tolerance can be attained by administering highor low doses of peptides according to the invention. The amount ofpeptide will depend on the route of administration, the time ofadministration, the age of the patient as well as general healthconditions and diet.

In general, a dosage of 0.01 to 10000 μg of peptide per kg body weight;preferably 0.05 to 500 μg, more preferably 0.1 to 100 μg of peptide canbe used.

Pharmaceutical acceptable carriers are well known to those skilled inthe art and include, for example, sterile saline, lactose, sucrose,calcium phosphate, gelatin, dextrin, agar, pectin, peanut oil, oliveoil, sesame oil and water. Other carriers may be, for example MHC classII molecules, if desired embedded in liposomes.

In addition the pharmaceutical composition according to the inventionmay comprise one or more adjuvants. Suitable adjuvants include, amongstothers, aluminum hydroxide, aluminum phosphate, amphigen, tocophenols,monophosphenyl lipid A, muramyl dipeptide and saponins such as Quill A.Prcferably, the adjuvants to be used in the tolerance therapy accordingto the invention are mucosal adjuvants such as the cholera toxineB-subunit or carbomers, which bind to the mucosal epithelium. The amountof adjuvant depends on the nature of the adjuvant itself.

Furthermore the pharmaceutical composition according to the inventionmay comprise one or more stabilizers such as, for example, carbohydratesincluding sorbitol, mannitol, starch, sucrosedextrin and glucose,proteins such as albumin or casein, and buffers like alkalinephosphates.

Suitable administration routes are e.g. intramuscular injections,subcutaneous injections, intravenous injections or intraperitonealinjections, oral administration and nasal administration such as sprays.

It is another object of the invention to provide a method for detectingautoreactive T cells involved in the destruction of articular cartilageand test kits to be used in said method. Thus, the peptides according tothe invention are also very suitable for use in a diagnostic method todetect the presence of activated autoreactive T cells involved in thechronic inflammation and destruction of the articular cartilage.

The diagnostic method according to the invention comprises the followingsteps:

-   -   a) isolation of the peripheral blood mononuclear cells (PBMC)        from a blood sample of an individual,    -   b) culture said PBMC under suitable conditions,    -   c) incubation of said PBMC culture in the presence of one or        more peptides according to the invention, and    -   d) detection of a response of T cells, for example a        proliferative response, indicating the presence of activated        autoreactive T cells in the individual.

The detection of a proliferative response of T cells can be detected by,for example, the incorporation of ³H-thymidine.

Also within the scope of the invention are test kits which comprise oneor more peptides according to the invention. These test kits aresuitable for use in a diagnostic method according to the invention.

The following examples are illustrative for the invention and should inno way be interpreted as limiting the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

FIG. 1 a, b, c. Cross reactivity of three, different, HC gp-39-specifichybridomas (8B12, 14G11, 20H5with YKL-39 (266-278).

-   -   (CVR0271B-HC gp39 (263-275), KV0432B=YKL-39 (266-278),        CC0332B=Chi (269-282) KV0431A=YKL-39 (262-274). HCDA.8B12.1D8,        14G11.1H7and 20H5.4F6.2F6 are HLA-DRB1*0401- restricted        hybridomas specific for HC gp-39 (263-275). Activation of T-cell        hybridomas is expressed as IL-2 production.

FIG. 2

FIG. 2 a, 2 b, 2 c In vivo tolerization with HC gp-39 (263-275) orYKL-39 (266-268)

-   -   Balb/c mice were tolerized by intranasal application of 50, 10        or 2 micrograms of HC gp-39 (263-275), SEQ ID NO: 3, (FIG. 2 b)        or YKL-39 (266-278) (FIG. 2 c) followed by immunization with HC        gp-39 (263-275). Mice that were pretreated with saline or that        were left untreated were included as controls (FIG. 2 a).

EXAMPLES Example 1 Alignment of Sequences

The human chondrocyte protein, YKL-39 shares significant sequenceidentity with HC gp-39 (YKL-40). Another homologue of HC gp-39 issecreted by human macrophages and is termed chitotriosidase (Boot etal., 1995). The sequences corresponding to RSFTLASSETGVG (HC gp-39(263-275), SEQ ID NO: 3) were identified as HSFTLASAETTVG (SEQ ID NO: 2)within the YKL-39 protein (266-278) and as RSFTLASSSDTRVG (SEQ ID NO: 4)within macrophage chitotriosidase (269-282) respectively (Table 1). Chi(269-282) contains the HLA-DRB1*0401 peptide binding motif which waspreviously used for selection of T-cell epitopes within proteins. Incontrast, the YKL-39 (266-278) peptide does not contain this motif. Allpeptides were synthesized.

TABLE 1 Alignment of the HC gp-39 (263-278) sequence with thecorresponding region in YKL-39 and macrophage Chitotriosidase. HCgp-39263-275 R S F T L A S S - E T G V G (SEQ ID NO: 3) YKL-39 266-278 H S FT L A S A - E T T V G (SEQ ID NO: 2) Chi (269-282) R S F T L A S S S D TR V G (SEQ ID NO: 4)

Example 2 Binding of Peptides to HLA-DRB1*0401

The peptides from example 1 were tested for binding theDRB1*0401-encoded molecules. HLA-DR4 (DRB1*0401) molecules were purifiedfrom the homozygous EBV-transformed human B lymphoblastoid cell linesHuly138IC2 and the competition peptide HLA-DR binding assay wasperformed basically as described by Verheijden et al., 1997. Theaffinity of a given peptide for binding DRB1*0401-encoded molecules wasrelated to competition with a marker peptide. This relative bindingaffinity was defined as the peptide concentration at which the signalwas reduced to 50% (IC50). The HA-F peptide is a positive control(Hemagglutinin 307-319; PKFVKQNTLKLAT; at position 309 Y is substitutedby F; SEQ ID NO: 5). The peptide is known to have a high affinity forDRB1*0401 molecules.

As expected, the Chi (269-282) peptide was found to bind with highaffinity to DRB1*0401 (see table 2). The YKL-39 (266-278) peptide, whichdoes not accommodate the effective DRB1*0401 peptide binding motif,bound with very high affinity to DR4 (B1*0401).

TABLE 2 Peptide binding to HLA-DRB1*0401-encoded molecules IC50 valuespeptide batch Exp.A Exp.B Exp.C YKL39(262-274) KV0431A 0.006 0.005 NDYKL39(266-278) KV432B 0.035 0.032 0.12 HCgp39(263-275) CVR271B ND 0.0080.038 Chi(269-282) CC0332B 0.053 0.11 0.16 HA-F AE0690A 0.20  0.14 0.20ND = not determined

Example 3 Stimulation T-cell Hybridomas

Hybridomas specific for HC gp-39 (263-275) were tested for recognitionof the corresponding sequences.

To test the cross reactivity of the 3 different, HC gp39-specifichybridoma cell lines with the YKL-39 or the chitotriosidase peptide,5×10⁴ hybridoma cells and 2×10⁵ irradiated (12000 RAD), EBV-transformedB cells carrying the DRB1*0401 specificity were incubated in 150 μlvolumes in wells of a round-bottomed microtiter plate. Peptide antigen(HC gp-39 (263-275), YKL-39 (266-278), chitotriosidase (269-282) or acontrol peptide) was added in 50 μl volumes to duplicate wells.Forty-eight hr later 100 μl of the culture supernatant was assayed forIL-2 production using a sandwich ELISA with Pharmingen antibodiesspecific for mouse IL-2.

It was found that the synthetic peptide YKL-39 (266-278) generated aresponse similar to HC gp-39 (263-275) whereas the Chi (269-282) did notgenerate a response. The data suggest that the three different TCRsutilized by three different hybridomas do not discriminate between HCgp-39 (263-275) or YKL-39 (268-278) when presented by DRB1*0401-encodedmolecules (FIGS. 1 a, b, c) but do discriminate between HC gp-39(263-275) and Chi (269-282). The data indicate that YKL-39 (266-278) isa mimicry epitope of HC gp-39 (263-275). (FIGS. 1 a,b,c).

Example 4 Recognition of YKL-39 (266-278) by PBMC

Peripheral blood mononuclear cells (PBMC) were isolated from heparinizedperipheral blood by standard centrifugation on Ficoll-Paque (Pharmacia,Uppsala, Sweden). Cells were suspended in wells of a 24 well plate in aconcentration of 5×10⁵ cells per ml. Cells were incubated in mediumalone or in the presence of 10 or 50 μg/ml peptide antigen (YKL-39(266-278)). Cultures were incubated for 6 days at 37° C. in a humidifiedatmosphere of 5% CO2. Cells were then suspended and 100 or 150 μlvolumes of medium was distributed in 4-fold in wells of a 96-wellroundbottomed plate. Cells were then pulsed with 0.5 μCi (1.85×10⁴ Bq)[3H]thymidine ([3H]TdR) and 18 hr later incorporated radioactivity wasmeasured. Results as shown in table 3 are expressed as stimulationindices (SI) (antigen-specific counts/background counts).

From Table 3a it can be concluded that the YKL-39 (266-278) epitope isreadily recognized in RA patients. Table 3b indicates that recognitionof YKL,39 (266-278)) by PBMC coincides with recognition of HC gp-39(263-275) and HC gp-39 and furthermore that recognition of YKL-39(266-278) is generally more pronounced than recognition of HC gp-39(263-275).

TABLE 3a Recognition of the YKL-39 (266-278) epitope by PBMC from RApatients. SI SI Donor typing 10 μg/ml 50 μg/ml 242-0.2 NR 0404/15 3 <2337-0.2 R 0401/02 19 58 338-0.1 NR  03/14 <2 <2 454-0 R 0401/ 9 9 456-0R ND 15 4 457-0 NR ND <2 <2 458-0 R ND 4 27 459-0 R ND <2 25 460-0 NR ND3 <2 SI = antigen-specific counts/background counts. SI ≧ 5 are regardedpositive R = responder, NR = non-responder

TABLE 3b Recognition of YKL-39 (266-278) coincides with recognition ofHC gp-39(263-275) and HC gp-39 protein. YKL-39(266-278) HCgp-39(263-275) HC gp-39 R/N SI SI SI SI SI SI Donor μg/ml 10 50 10 50 1050 169 R 27 32 10 27 24 44 455 R 20 35 1 15 45 95 447 NR 1 2 1 1 1 1 327R 6 5 3 5 12 19 SI = antigen-specific counts/background counts. SI ≧ 5are regarded positive. R = responder, NR = non-responder. NT = nottested. Donor 447 responds to Tetanus toxoid and Candida Albicans.

Example 5 Tolerance Induction

A HC gp-39 (263-275)-specific DTH assay suitable to monitor toleranceinduction with peptide antigens was developed. Immunisation of Balb/cmice with HC gp-39 (263-275) in incomplete Freunds adjuvant (IFA) wasfound to be effective in the induction of a DTH response followingchallenge with the HC gp-39 (263-275) peptide. This peptide-based DTHsystem was used to detect modulation of the DTH response by nasalapplication of HC gp-39 (263-275) peptide. It was found that nasalapplication of HC gp-39 (263-275), in a dose-dependent manner,downmodulated the HC gp-39 (263-275) induced DTH response. Nasalapplication of YKL-39 (266-278), however, resulted in a more enhanceddownmodulation of the DTH response, indicating that YKL-39 (266-278) canefficiently tolerize a peptide-specific response induced with HC gp-39(263-275) (Table 4, FIG. 2 a,b,c).

TABLE 4 Experimental set-up tolerization experiment toler- Pretreatmentsensibilisation challenge ance none HC gp-39 (263-275) HC gp-39(263-275) no saline HC gp-39 (263-275) HC gp-39 (263-275) no HC gp-39(263-275) HC gp-39 (263-275) HC gp-39 (263-275) yes YKL-39 (266-278) HCgp-39 (263-275) HC gp-39 (263-275) yes

1. An isolated peptide of 9-55 amino acids comprising the amino acidsequence FTLASAETT (SEQ ID NO: 1).
 2. An isolated peptide of 13-55 aminoacids comprising the amino acid sequence HSFTLASAETTVG (SEQ ID NO: 2).3. The peptide according to claim 1 having an amino acid sequence of 9to 25 amino acids.
 4. The peptide according to claim 1 consistingessentially of the amino acid sequence FTLASAETT (SEQ ID NO: 1) orHSFTLASAETTVG (SEQ ID NO: 2).
 5. Pharmaceutical composition comprisingat least one peptide according to claim 1 and a pharmaceuticallyacceptable carrier.
 6. A method for the manufacture of a pharmaceuticalpreparation for the induction of specific T-cell tolerance to anautoantigen in patients suffering from autoimmune disorders, comprisingcombining a peptide according to claim 1 with a pharmaceuticallyacceptable carrier.
 7. Diagnostic composition comprising at least onepeptide according to claim 1 and a detection agent.
 8. The peptideaccording to claim 2 having an amino acid sequence of 13 to 25 aminoacids.
 9. A method for detecting autoreactive T-cells comprising:providing peripheral blood mononuclear cells, culturing the peripheralblood mononuclear cells; incubating the peripheral blood mononuclearcells with at least one peptide according to claim 1; and detecting areeponse of the peripheral blood mononuclear cells to the presence ofthe peptide.